Acta Med Scand 203: 125-130, 1978
Platelet Aggregation in Diabetes Mellitus Hans Draminsky Petersen and Johs. Gormsen From Department T,Bispebjerg Hospital, and the Coagulation Laboratory, Municipal Hospital, Copenhagen, Denmark
ABSTRACT. Thirty-eight patients with juvenile diabetes mellitus, aged 21-77, were tested for platelet aggregation in vitro. Vascular complications were found in 20 patients and diabetic retinopathy in 16 of these. All patients received their usual dose of insulin in the morning on the day of the examinations, which were carried out shortly before or 2-3 hours after lunch. Ninety normal controls were tested at the same time of day. The aggregation was estimated turbidometrically and defined by the threshold concentrationof adenosinediphosphateor adrenaline that produced a secondary aggregation with a light transmission not less than 80% of that given by the platelet-poor plasma. No significant differences could be demonstrated in platelet aggregation between the normal controls and the patients with diabetes mellitus or any subgroup of these. No correlation was found between the threshold concentrations and the plasma levels of glucose or &hydroxybutyric acid.
Many studies have been performed in recent years to illustrate the significance of platelets in the development of arterial thromboatherosclerosis. Shortened platelet survival time and/or some kind of platelet hyperactivity have been found in patients with transient cerebral ischemia (TCI), ischemic heart disease (IHD), or peripheral thromboatherosclerosis (PTA) (22, 31). If platelets are of any significance for the above disorders, they might also be involved in the development of diabetic macro- and microangiopathy (21). The conclusions from studies in diabetic patients are contradictory. Some authors report normal aggregation (13, 16), some find hyperactive platelets (7, 19, 2 3 , and some believe that any increased platelet activity is due to a plasma factor (18).
The aim of the present work has been to study the platelet aggregation in vitro in patients with diabetes, using a technique that has proved valuable in other studies, in which we have demonstrated a distinct increase in adenosine diphosphate (ADP)induced aggregation in patients suffering from TCI (2), IHD, and PTA (1 l), and in patients recovering after acute cerebral infarction (20). The patients have been examined after taking their insulin and meals as usual. The normal controls were tested at the same time of day, likewise without fasting. PLATELET AGGREGATION STUDIES Platelet aggregation was evaluated turbidometrically according to the principles described by Born (4). The experiments were performed in a Payton aggregometer model 300 (stirring at 900 r.p.m.) at 37°C. Blood was drawn by puncture of a cubital vein after slight compression, 9 parts of blood being kept in a plastic tube with 1 part of trisodium citrate 3.13%. Platelet-rich and platelet-poor plasma was obtained by immediate centrifugation at room temperature at 200xg for 3 min and 1Oooxg for 20 min, respectively. The plasma was pipetted by plastic material into other plastic tubes, kept capped at room temperature for not more than one hour and prewarmed at 37°C for 3 min before testing. The plateletrich plasma contained 2W35OOoo plateletslpl, counted in a Thomas’ counting chamber. Platelet aggregation was started by adding ADP (Sigma Chemical Co., St. Louis, USA) in 0.05 ml of sterile saline to 0.5 ml platelet-rich plasma. The final concentrations of ADP were 0.25,0.50, 1.0, 2.0 and 5.0 pM. Platelet aggregation was also started by adding adrenaline (Gauche, RhBne-Poulenc, Paris), giving final concentrations of 0.01,0.05,0.5 and 1.0 pg/ml. Estimates were made of the threshold concentration, which is the lowest concentration of ADP or adrenaline that is able to produce a secondary aggregation with an amplitude corresponding to not less than 80% of that obtained with the transmission given by the platelet-poor plasma (1 1). Acta Med Scand 203
H . Draminsky Petersen and J . Gormsen
0.25
1
0.50
1
I
2
5
1
>5
I I I , 0 NORMAL
I
DIAB
Fig. I . Threshold concentration of ADP ( p M )in normal controls and in patients with diabetes rnellitus.
Other methods The concentrations in plasma of Hb, glucose, creatinine, and P-hydroxybutyric acid were estimated by the Department of Clinical Chemistry, Bispebjerg Hospital, which also performed other routine tests.
STUDY POPULATION All individuals tested were carefully questioned about any intake of drugs, which might influence platelet aggregation, and were excluded if they had taken such drugs during the past week. The control material comprises 90 persons, 20-70 years old, all donors at the Blood Bank of the Municipal Hospital of Copenhagen. Most samples were taken between 10 a.m. and 2 p.m. The patient group comprises 38 patients (21 men and 17 women), 21-77 years old, with juvenile diabetes mellitus. All patients were treated with insulin, most with Insulin Retard@(Leo), which was given as usual in the morning on the day of the examination. Most of the patients were Acla Med Scand 203
examined between 1 and 4 p.m., a few before noon, and they were not fasting. Diagnosis. Diabetic retinopathy was diagnosed on the basis of ophthalmologic criteria defined by one of the authors (H.P.D.). The diagnosis of nephropathy, was based on persisting proteinuria, or elevated serum creatinine, for more than one year, and the diagnoses of atherosclerosis and diabetic neuropathy, were based on clinical examination and ECG. Eighteen of the 38 patients were without any vascular complications according to our definition. Of the 20 patients with vascular complications, diabetic retinopathy was found in 16, of whom 5 had nephropathy and 10 neuropathy, and distinct arterial insufficiency in 6.
RESULTS Platelet aggregation studies The threshold concentrations of ADP and adrenaline in the controls and the diabetic patients are given in Figs. 1 and 2. The threshold concentration of ADY was < 1 pM in 33 % of the controls and in 21 %
Platelet aggregation in diabetes mellitus
127
Fig. 2. Threshold concentration of adrenaline in plasma (pg/ml)in normal controls and in patients with diabetes mellitus.
of the patients. The threshold concentration of adrenaline was cO.1 pglml in 40% of the controls and 27% of the patients. Accordingly, no hyperaggregability was found in the patients with this technique. No significant differences were detected in the threshold concentrations of either ADP or adrenaline between the control group and the diabetic group (Figs. 3 and 4), nor between the control group and any subgroup of patients with diabetic complications. No correlation was found between the threshold concentration and blood levels of glucose or phydroxybutyric acid. The correlationbetween threshold concentrations of ADP and adrenaline was identical in the controls and the diabetics. The differences between light transmission in platelet-rich and platelet-poor plasma were identical in both groups and the results were not due to hyperlipidemia.
DISCUSSION Abnormalities in the hemostatic balance might be of some significance for the development of diabetic macro- and microangiopathy (21). AlmCr and Pandolfi (1) found a reduced fibrinolytic activity and Pandolfi et al. (24) an increased von Willebrand factor activity in patients with diabetic retinopathy. Platelet thrombi were demonstrated by Fagerberg (9) in the small vessels of sural nerve biopsy in diabetic neuropathy, by Timperley et al. (28) in the cerebral microcirculation in patients dying from ketoacidosis, and by Bloodworth and Monitor (3) in the retinal vessels in experimental canine diabetic retinopathy. Ferguson et al. (10) showed increased platelet and fibrinogen turnover in patients with diabetes mellitus. Studies on platelet aggregation in vivo and in vitro in diabetic patients have been carried out for years. They have been performed in the morning on fasting patients. Heath et al. (16) found an increased Acfa Med Scand 203
128
H . Draminsky Petersen and J . Gormsen
1I.C
\
t
h
%
lu P \
s 4
Q
O"
Q
P
0.05
Fig. 3. Correlation between ADP and adrenaline thresh-
0.01
< 0.01
9
0.5
1.0
2.0
pM
3 .O ADP
aggregation tendency in vitro, defined by a broader amplitude after adding ADP in low concentrations (1-2 pM) and by a decrease in disaggregation in patients with severe diabetic retinopathy. Hassanein et al. (15) found decreased disaggregation after addition of ADP in diabetics, unrelated to vascular complications. An i.v. injection of insulin was followed by an increase in aggregation rates. The maximal amplitude, however, was reduced and the disaggregation increased. The decrease in the primary disaggregation was especially marked in patients with high cholesterol levels. Kwaan et al. (18) showed that ADP-induced (but not adrenalineinduced) aggregation of normal platelet-rich plasma was increased by adding plasma from patients with diabetes, especially if the diabetics had vascular complications. They found no relationship between the increase in aggregation tendency and age or weight, dose of insulin, and levels of blood glucose, triglyceride, cholesterol, free fatty acids or albumin. They also noted that aggregation was not affected by sulfonylurea, i.v. glucagon or insulin. Acta Med Scand 203
4.0
15.0
old concentrations in normal controls.
Neither did aspirin affect the aggregation tendency. Sage1 et al. (26) found an increased aggregation tendency, determined as an increase in light transmission after 4 min, using low concentrations of ADP (0.125 and 0.25 pM) or low concentrations of adrenaline and collagen in platelet-rich plasma from diabetics, unrelated to presence of vascular complications. There was no relation between fasting blood glucose and the aggregation tendency. The increase was reduced by aspirin, i.v. tolbutamide or peroral glucose, whereas sulfonylurea perorally had no effect. OMalley et al. (23) found lower threshold concentrations of ADP and adrenaline in 20 patients with diabetic peripheral neuropathy, but no significant differences between the controls and the diabetic group without vascular complications. They observed, like Breddin et al. ( 5 ) and Wu and Hoak (30), an increased tendency to formation of spontaneous platelet aggregates in vivo or in vitro. Colwell et al. (7) demonstrated an increased sensitivity to ADP and adrenaline, unrelated to vascular complications but related to increased von Willebrand
Platelet aggregation in diabetes mellitus
11.0
129
k-
4
< 1
h
%
lu
3 4
s Q
O"
a 9
0.0:
Fig. 4. Correlation between
0.0I
ADP and adrenaline thresh-
< QOI
old concentrations in pa-
C
I
pM
L!
tients with diabetes mellitus.
ADP
factor activity and increased blood concentrations of growth hormone. They also demonstrated that diabetic platelets produced more PGE,-like material than normal platelets (14). Passa et al. (25) normalized the increased ADP aggregation in diabetic retinopathy by hypophysectomy. The reports are accordingly rather conflicting. Also some authors (13) have not been able to find significant changes in platelet aggregations in patients with diabetes mellitus. We have previously demonstrated a significant increase in ADP-induced platelet aggregation, as defined by lower threshold concentrations in patients with TCI (2), IHD, and PTA (11) and after acute cerebral infarction (20). We applied the same technique in the present study in diabetic patients. All our patients suffered from juvenile diabetes mellitus, 20 with vascular complications, and 16 of these had diabetic retinopathy. Patients were tested during the day, not fasting, having taken their usual dose of insulin. Normal controls were tested at the same time of day, also not fasting. 9- 78298I
4.0
With this procedure we found that the aggregation tendency was not increased as defined by our criteria. The threshold concentrations of ADP and adrenaline were not lower in the diabetic group, whether vascular complications were present or not. The amplitude will be reduced in cases of hyperlipidemia, but this was not the reason for the lack of a demonstrable increase in our study. We found no correlation between aggregation tendency and plasma levels of glucose, p-hydroxybutyric acid, factors illustrating the regulation of the metabolism. Diurnal fluctuations in aggregation, if present, could hardly be due to fluctuations in the plasma concentrations of glucose, potassium, lipoproteins, corticosteroids, glucagon or growth hormone (6, 11, 12, 29). We found that an i.v. infusion of somatostatin did not influence platelet aggregation (8). Changes in levels of catecholamines during hyper- or hypoglycemia might influence the aggregability. Whether changes in the plasma level of the physiological aggregation inhibitor, 2,3-diphosphoActa Med Scand 203
130
H . Draminsky Petersen and J . Gormsen
glycerate, the concentration of which is increased in diabetic plasma (17), may influence our results, is also unknown t o us a t present.
ACKNOWLEDGEMENTS Supported by grants from the Danish Heart Foundation and the Danish Hospital Foundation for Medical Research, Region of Copenhagen, Faroe Islands and Greenland.
REFERENCES I . Almtr, L. 0. & Pandolfi, M.: Fibrinolysis and diabetic retinopathy. Diabetes (Suppl) 2: 80, 1976. 2. Andersen, L. A. & Gormsen, J.: Platelet aggregation and fibrinolytic activity in transient cerebral ischemia. Acta Neurol Scand 55: 76, 1976. 3. Bloodworth, I. M. B. & Monitor, D. L.: Ultrastructurd aspects of human and canine diabetic retinopathy. Invest Ophthalmol4: 1037, 1965. 4. Born, G. V. R.: Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 194: 927, 1%2. 5 . Breddin, K., Grun, H., Krzywanek, H. J. & Schremmer, W. P.: On the measurement of spontaneous platelet aggregation. The platelet aggregation test. 111. Thromb Haemostas (Stuttg) 34: 666. 1976. 6. Carvalho, A. C., Colman, R. W. & Lees, R. S.: Platelet function in hyperlipoproteinemia. N Engl J Med 290: 434, 1974. 7. Colwell, J. A., Halushka, P. V., Sargi, K., Levine, J., Sagel, J. & Nair, R. M. G.: Altered platelet function in diabetes mellitus. Diabetes (Suppl) 11: 826, 1976. 8. Faber, J., Gormsen, J., Friis, T., Kirkegaard, C., Birk Lauridsen, U., Nerup, J., Rogowski, P. & SiersbaekNielsen, K.: Effect of Somatostatin on TSH-induced T,-release and on platelet function in normal man. Horm Metab Res 9: 165, 1977. 9. Fagerberg, S. E.: Diabetic neuropathy a clinical and historical study on the significance of vascular affections. Acta Med Scand (Suppl) 345, 1959. 10. Ferguson, J. C., Markay, N., Philip, I. A. D. & Summer, D. J.: Platelet and fibrinogen kinetic studies in diabetes mellitus (Abstract). Br J Haematol 25: 545, 1973. 11. Gormsen, J., Nielsen, J. D. & Andersen, L. A.: ADPinduced platelet aggregation in vitro in patients with ischemic heart disease and peripheral thromboatherosclerosis. Acta Med Scand 201: 509, 1977 12. Guerra, S. M. 0. & Kitabchi, A. E.: Comparison of the effectiveness of various routes of insulin injection: Insulin levels and glucose response in normal subjects. J Clin Endocrinol Metab 42: 869, 1976. 13. Haanen, C. & Holdrinet, A.: Blood platelet aggregation under various conditions and in some diseases. Exp Biol Med 3: 164, 1%8. 14. Halushka, P. V., Weiser, C., Chambers, A. & Colwell, J.: Synthesis of prostaglandin “E like” material (PGE) in diabetic and normal platelets. In: Advance Acta
Med Scand 203
in prostaglandin and thromboxane research (ed. B. Samuelsson & R. Paolette), p. 853. Raven Press, New York 1976. 15. Hassanein, A. A., El-Gad, Th. & El-Baz, S.: Platelet aggregation in diabetes mellitus and the effect of insulin in vivo on aggregation. Thromb Diath Haemorrh 27: 114, 1972. 16. Heath, H., Brigden, W. D., Canever, J. V., Pollock, J., Hunter, P. R., Kelsey, J. & Bloom, A.: Platelet adhesiveness and aggregation in relation to diabetic retinopathy. Diabetologia 7: 308, 1971. 17. Iatridis, S. G., Iatridis, P. G., Markidou, S. G. & Ratz, B. H.: 2,3-Diphosphoglycerate: a physiological inhibitor of platelet aggregation. Science 87: 259, 1975. 18. Kwaan, H. C., Colwell, J. A., Cruz, S., Suwanwela, N. & Dobbie, J. G.: Increased platelet aggregation in diabetes mellitus. J Lab Clin Med 80: 236, 1972. 19. Leone, G., Bizzi, B., Accorra, F. & Boni, P.: Functional aspects of platelets in diabetes mellitus. In: Platelet aggregation and drugs (ed. L. Caprino & F. C. Rossi), pp. 49-61. Academic Press, New York 1974. 20. Lou, H. C., Dalsgard-Nielsen, J., Bomholt, A. & Gormsen, J.: Platelet hyperaggregability in young patients with completed stroke. Neurology. In preparation. 21. McMillan, D.E.: Deterioration of the microcirculation in diabetes mellitus. Diabetes 24: 944, 1975. 22. Mustard, J. F. & Packham, M. A.: The role of blood and platelets in atherosclerosis and the complications of atherosclerosis. Thromb Diath Haemorrh 33: 444, 1975. 23. OMalley, B. C., Timperley, W. R., Ward, J. D. & Porter, N. R.: Platelet abnormalities in diabetic peripheral neuropathy. Lancet 2: 1274, 1975. 24. Pandolfi, M., AlmCr, L. A. & Holmberg, L.: Increased von Willebrand-antihaemophilicfactor A in diabetic retinopathy. Acta Ophthalmol 52: 823, 1974. 25. Passa, P., Bensossan, D., Levy-Toledano, S., Caen, J. & Canivet, J.: Etude de I’aggregation plaquettaire en course de la retinopathie diabttique. Atherosclerosis 19: 277, 1974. 26. Sagel, J., Colwell, J. A., Crook, L. & Laimins, M.: Increased platelet aggregation in early diabetes mellitus. Ann Intern Med 82: 733, 1975. 27. Sano, T., Boxer, M. G. J., Boxer, L. A. & Yokoyama, M.: Platelet sensitivity to aggregation in normal and diseased groups. Thromb Diath Haemorrh 25: 524, 1971. 28. Timperley, W. R., Preston, F. E. & Ward, J. D.: Cerebral intravascular coagulation in diabetic ketoacidosis. Lancet 1: 952, 1974. 29. Willey, J. S., Kuchibhotla, J., Shaller, C. C. & Colman, R. W.: Potassium uptake and release by human platelets. Blood 48: 185, 1976. 30. Wu, K. K. & Hoak, J. C.: Spontaneous platelet aggregation in arterial insufficiency: mechanism and implications. Thromb Haemostas (Stuttg) 35: 707, 1976. 31. Yamazaki, H., Takahashi, T. & Sano, T.: Hyperaggregability of platelets in thromboembolic disorders. Thromb Diath Haemorrh 34: 94, 1975.
Platelet Aggregation in Diabetes Mellitus Hans Draminsky Petersen and Johs. Gormsen From Department T,Bispebjerg Hospital, and the Coagulation Laboratory, Municipal Hospital, Copenhagen, Denmark
ABSTRACT. Thirty-eight patients with juvenile diabetes mellitus, aged 21-77, were tested for platelet aggregation in vitro. Vascular complications were found in 20 patients and diabetic retinopathy in 16 of these. All patients received their usual dose of insulin in the morning on the day of the examinations, which were carried out shortly before or 2-3 hours after lunch. Ninety normal controls were tested at the same time of day. The aggregation was estimated turbidometrically and defined by the threshold concentrationof adenosinediphosphateor adrenaline that produced a secondary aggregation with a light transmission not less than 80% of that given by the platelet-poor plasma. No significant differences could be demonstrated in platelet aggregation between the normal controls and the patients with diabetes mellitus or any subgroup of these. No correlation was found between the threshold concentrations and the plasma levels of glucose or &hydroxybutyric acid.
Many studies have been performed in recent years to illustrate the significance of platelets in the development of arterial thromboatherosclerosis. Shortened platelet survival time and/or some kind of platelet hyperactivity have been found in patients with transient cerebral ischemia (TCI), ischemic heart disease (IHD), or peripheral thromboatherosclerosis (PTA) (22, 31). If platelets are of any significance for the above disorders, they might also be involved in the development of diabetic macro- and microangiopathy (21). The conclusions from studies in diabetic patients are contradictory. Some authors report normal aggregation (13, 16), some find hyperactive platelets (7, 19, 2 3 , and some believe that any increased platelet activity is due to a plasma factor (18).
The aim of the present work has been to study the platelet aggregation in vitro in patients with diabetes, using a technique that has proved valuable in other studies, in which we have demonstrated a distinct increase in adenosine diphosphate (ADP)induced aggregation in patients suffering from TCI (2), IHD, and PTA (1 l), and in patients recovering after acute cerebral infarction (20). The patients have been examined after taking their insulin and meals as usual. The normal controls were tested at the same time of day, likewise without fasting. PLATELET AGGREGATION STUDIES Platelet aggregation was evaluated turbidometrically according to the principles described by Born (4). The experiments were performed in a Payton aggregometer model 300 (stirring at 900 r.p.m.) at 37°C. Blood was drawn by puncture of a cubital vein after slight compression, 9 parts of blood being kept in a plastic tube with 1 part of trisodium citrate 3.13%. Platelet-rich and platelet-poor plasma was obtained by immediate centrifugation at room temperature at 200xg for 3 min and 1Oooxg for 20 min, respectively. The plasma was pipetted by plastic material into other plastic tubes, kept capped at room temperature for not more than one hour and prewarmed at 37°C for 3 min before testing. The plateletrich plasma contained 2W35OOoo plateletslpl, counted in a Thomas’ counting chamber. Platelet aggregation was started by adding ADP (Sigma Chemical Co., St. Louis, USA) in 0.05 ml of sterile saline to 0.5 ml platelet-rich plasma. The final concentrations of ADP were 0.25,0.50, 1.0, 2.0 and 5.0 pM. Platelet aggregation was also started by adding adrenaline (Gauche, RhBne-Poulenc, Paris), giving final concentrations of 0.01,0.05,0.5 and 1.0 pg/ml. Estimates were made of the threshold concentration, which is the lowest concentration of ADP or adrenaline that is able to produce a secondary aggregation with an amplitude corresponding to not less than 80% of that obtained with the transmission given by the platelet-poor plasma (1 1). Acta Med Scand 203
H . Draminsky Petersen and J . Gormsen
0.25
1
0.50
1
I
2
5
1
>5
I I I , 0 NORMAL
I
DIAB
Fig. I . Threshold concentration of ADP ( p M )in normal controls and in patients with diabetes rnellitus.
Other methods The concentrations in plasma of Hb, glucose, creatinine, and P-hydroxybutyric acid were estimated by the Department of Clinical Chemistry, Bispebjerg Hospital, which also performed other routine tests.
STUDY POPULATION All individuals tested were carefully questioned about any intake of drugs, which might influence platelet aggregation, and were excluded if they had taken such drugs during the past week. The control material comprises 90 persons, 20-70 years old, all donors at the Blood Bank of the Municipal Hospital of Copenhagen. Most samples were taken between 10 a.m. and 2 p.m. The patient group comprises 38 patients (21 men and 17 women), 21-77 years old, with juvenile diabetes mellitus. All patients were treated with insulin, most with Insulin Retard@(Leo), which was given as usual in the morning on the day of the examination. Most of the patients were Acla Med Scand 203
examined between 1 and 4 p.m., a few before noon, and they were not fasting. Diagnosis. Diabetic retinopathy was diagnosed on the basis of ophthalmologic criteria defined by one of the authors (H.P.D.). The diagnosis of nephropathy, was based on persisting proteinuria, or elevated serum creatinine, for more than one year, and the diagnoses of atherosclerosis and diabetic neuropathy, were based on clinical examination and ECG. Eighteen of the 38 patients were without any vascular complications according to our definition. Of the 20 patients with vascular complications, diabetic retinopathy was found in 16, of whom 5 had nephropathy and 10 neuropathy, and distinct arterial insufficiency in 6.
RESULTS Platelet aggregation studies The threshold concentrations of ADP and adrenaline in the controls and the diabetic patients are given in Figs. 1 and 2. The threshold concentration of ADY was < 1 pM in 33 % of the controls and in 21 %
Platelet aggregation in diabetes mellitus
127
Fig. 2. Threshold concentration of adrenaline in plasma (pg/ml)in normal controls and in patients with diabetes mellitus.
of the patients. The threshold concentration of adrenaline was cO.1 pglml in 40% of the controls and 27% of the patients. Accordingly, no hyperaggregability was found in the patients with this technique. No significant differences were detected in the threshold concentrations of either ADP or adrenaline between the control group and the diabetic group (Figs. 3 and 4), nor between the control group and any subgroup of patients with diabetic complications. No correlation was found between the threshold concentration and blood levels of glucose or phydroxybutyric acid. The correlationbetween threshold concentrations of ADP and adrenaline was identical in the controls and the diabetics. The differences between light transmission in platelet-rich and platelet-poor plasma were identical in both groups and the results were not due to hyperlipidemia.
DISCUSSION Abnormalities in the hemostatic balance might be of some significance for the development of diabetic macro- and microangiopathy (21). AlmCr and Pandolfi (1) found a reduced fibrinolytic activity and Pandolfi et al. (24) an increased von Willebrand factor activity in patients with diabetic retinopathy. Platelet thrombi were demonstrated by Fagerberg (9) in the small vessels of sural nerve biopsy in diabetic neuropathy, by Timperley et al. (28) in the cerebral microcirculation in patients dying from ketoacidosis, and by Bloodworth and Monitor (3) in the retinal vessels in experimental canine diabetic retinopathy. Ferguson et al. (10) showed increased platelet and fibrinogen turnover in patients with diabetes mellitus. Studies on platelet aggregation in vivo and in vitro in diabetic patients have been carried out for years. They have been performed in the morning on fasting patients. Heath et al. (16) found an increased Acfa Med Scand 203
128
H . Draminsky Petersen and J . Gormsen
1I.C
\
t
h
%
lu P \
s 4
Q
O"
Q
P
0.05
Fig. 3. Correlation between ADP and adrenaline thresh-
0.01
< 0.01
9
0.5
1.0
2.0
pM
3 .O ADP
aggregation tendency in vitro, defined by a broader amplitude after adding ADP in low concentrations (1-2 pM) and by a decrease in disaggregation in patients with severe diabetic retinopathy. Hassanein et al. (15) found decreased disaggregation after addition of ADP in diabetics, unrelated to vascular complications. An i.v. injection of insulin was followed by an increase in aggregation rates. The maximal amplitude, however, was reduced and the disaggregation increased. The decrease in the primary disaggregation was especially marked in patients with high cholesterol levels. Kwaan et al. (18) showed that ADP-induced (but not adrenalineinduced) aggregation of normal platelet-rich plasma was increased by adding plasma from patients with diabetes, especially if the diabetics had vascular complications. They found no relationship between the increase in aggregation tendency and age or weight, dose of insulin, and levels of blood glucose, triglyceride, cholesterol, free fatty acids or albumin. They also noted that aggregation was not affected by sulfonylurea, i.v. glucagon or insulin. Acta Med Scand 203
4.0
15.0
old concentrations in normal controls.
Neither did aspirin affect the aggregation tendency. Sage1 et al. (26) found an increased aggregation tendency, determined as an increase in light transmission after 4 min, using low concentrations of ADP (0.125 and 0.25 pM) or low concentrations of adrenaline and collagen in platelet-rich plasma from diabetics, unrelated to presence of vascular complications. There was no relation between fasting blood glucose and the aggregation tendency. The increase was reduced by aspirin, i.v. tolbutamide or peroral glucose, whereas sulfonylurea perorally had no effect. OMalley et al. (23) found lower threshold concentrations of ADP and adrenaline in 20 patients with diabetic peripheral neuropathy, but no significant differences between the controls and the diabetic group without vascular complications. They observed, like Breddin et al. ( 5 ) and Wu and Hoak (30), an increased tendency to formation of spontaneous platelet aggregates in vivo or in vitro. Colwell et al. (7) demonstrated an increased sensitivity to ADP and adrenaline, unrelated to vascular complications but related to increased von Willebrand
Platelet aggregation in diabetes mellitus
11.0
129
k-
4
< 1
h
%
lu
3 4
s Q
O"
a 9
0.0:
Fig. 4. Correlation between
0.0I
ADP and adrenaline thresh-
< QOI
old concentrations in pa-
C
I
pM
L!
tients with diabetes mellitus.
ADP
factor activity and increased blood concentrations of growth hormone. They also demonstrated that diabetic platelets produced more PGE,-like material than normal platelets (14). Passa et al. (25) normalized the increased ADP aggregation in diabetic retinopathy by hypophysectomy. The reports are accordingly rather conflicting. Also some authors (13) have not been able to find significant changes in platelet aggregations in patients with diabetes mellitus. We have previously demonstrated a significant increase in ADP-induced platelet aggregation, as defined by lower threshold concentrations in patients with TCI (2), IHD, and PTA (11) and after acute cerebral infarction (20). We applied the same technique in the present study in diabetic patients. All our patients suffered from juvenile diabetes mellitus, 20 with vascular complications, and 16 of these had diabetic retinopathy. Patients were tested during the day, not fasting, having taken their usual dose of insulin. Normal controls were tested at the same time of day, also not fasting. 9- 78298I
4.0
With this procedure we found that the aggregation tendency was not increased as defined by our criteria. The threshold concentrations of ADP and adrenaline were not lower in the diabetic group, whether vascular complications were present or not. The amplitude will be reduced in cases of hyperlipidemia, but this was not the reason for the lack of a demonstrable increase in our study. We found no correlation between aggregation tendency and plasma levels of glucose, p-hydroxybutyric acid, factors illustrating the regulation of the metabolism. Diurnal fluctuations in aggregation, if present, could hardly be due to fluctuations in the plasma concentrations of glucose, potassium, lipoproteins, corticosteroids, glucagon or growth hormone (6, 11, 12, 29). We found that an i.v. infusion of somatostatin did not influence platelet aggregation (8). Changes in levels of catecholamines during hyper- or hypoglycemia might influence the aggregability. Whether changes in the plasma level of the physiological aggregation inhibitor, 2,3-diphosphoActa Med Scand 203
130
H . Draminsky Petersen and J . Gormsen
glycerate, the concentration of which is increased in diabetic plasma (17), may influence our results, is also unknown t o us a t present.
ACKNOWLEDGEMENTS Supported by grants from the Danish Heart Foundation and the Danish Hospital Foundation for Medical Research, Region of Copenhagen, Faroe Islands and Greenland.
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